• Proceedings of the Korean Society of Plant Pathology Conference
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• 2003.10a
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• pp.69.1-69
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• 2003

RSH (relA/spoT homolog) has been known to determine the level of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which are the effector nucleotide of the prokaryotic stringent response and also play a role in antibiotic production and differentiation in Streptomyces species but not a little in eukaryotic organism, especially in plant. Salicylic acid (SA), a critical signal molecule of establishing systemic acquired resistance (SAR), could induce SAR in Pepper (Capcicum annuum) against Phytophthora capsici. And the extent of SAR induction was in proportion to the dosage of SA (or BTH). Suppression subtractive hybridization (SSH), a PCR-based method for cDNA subtraction, was carried out between SA-treated and non-SA-treated pepper leaves to isolate genes which may be responsible for defense signaling against pathogens. Early upregulated gene was selected from reverse northern and kinetics of SSH-genes transcripts in SA-treated pepper leaves upon SA treatment. Full-length cDNA of the gene (PepRSH； Pepper RelA / SpoT homolog) had an open reading frame (ORF) of 2166 bp encoding a protein of 722 amino acids and a significant homology with (p)ppGpp phosphohydrolase or synthetase. Genomic DNA gel blot analysis showed that pepper genome has at least single copy of PepRSH. PepRSH transcripts was very low in untreated pepper leaves but strongly induced by SA and methyljasmonic acid (MeJA), indicating that PepRSH may share common SA and MeJA-mediated signal transduction pathway Functional analysis in E. coli showed PepRSH confers phenotypes associated with (p)ppGpp synthesis through a complementation using active site mutagenesis.

• Proceedings of the Korean Society for Bioinformatics Conference
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• 2005.09a
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• pp.139-143
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• 2005

Cytochrome P450 14${\alpha}$-sterol demethylase enzyme (CYP51) is the target a of azole type antifungals. The azole blocks the ergosterol synthesis and thereby inhibits fungal growth. A three-dimensional (3D) homology model of CYP51 from Candida albicans was constructed based on the X-ray crystal structure of CYP51 from Mycobacterium tuberculosis. Using this model, the binding modes for the substrate (24-methylene-24, 25-dihydrolanosterol) and the known inhibitors (fluconazole, voriconazole, oxiconazole, miconazole) were predicted from docking. Virtual screening was performed employing Structure Based Focusing (SBF). In this procedure, the pharmacophore models for database search were generated from the protein-ligands interactions each other. The initial structure-based virtual screening selected 15 compounds from a commercial available 3D database of approximately 50,000 molecule library, Being evaluated by a cell-based assay, 5 compounds were further identified as the potent inhibitors of Candida albicans CYP51 (CACYP51) with low minimal inhibitory concentration (MIC) range. BMD-09-01${\sim}$BMD-09-04 MIC range was 0.5 ${\mu}$g/ml and BMD-09-05 was 1 ${\mu}$g/ml. These new inhibitors provide a basis for some non-azole antifungal rational design of new, and more efficacious antifungal agents.

• Journal of Microbiology and Biotechnology
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• v.16 no.7
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• pp.1104-1110
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• 2006

Recombinant antibody-toxin is a bifunctional protein that binds and kills a target cell expressing a specific antigen on the surface of the cell, and its structure is chimeric, in which a toxin is fused to an antigen-binding domain such as scFv or Fab. Divalent antibody-toxin molecules showed higher cytotoxicities against cancer cell lines than monovalent molecules. However, the yields of the divalent molecules were very low. In this study, we introduced the CH2, CH3, or CH2-CH3 (=Fc) domain of antibody in the middle of the Fab-toxin between the hinge region of human IgG1 and the toxin domain to increase the yield. The covalently bonded dimer could be formed by three disulfide bridges from cysteine residues in the hinge region. The molecule with the CH3 domain showed about 3-fold higher dimerization yield than previously constructed Fab-toxin molecules, while maintaining the cytotoxic activity comparable to that of scFv-toxin. However, the introduction of CH2 or Fc domain to the same position showed little effect on the dimerization yield. We also observed that the introduction of the CH3 region made it possible to form noncovalently associated dimer molecules.

• Environmental Analysis Health and Toxicology
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• v.15 no.3
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• pp.69-80
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• 2000

ADP-rubosylation may be involved in the process of macrophage activation. Nitric oxide (NO) has emerged as an important intracellular and interacellular regulatory molecule with function as diverse as vasodilation, neural communication or host defense. NO is derived from the oxidation of the terminal guanidino nitrogen atom of L-arginine by the NADPH -dependent enzyme, nitric oxide synthase (NOS) which is one of the three different isomers in mammalian tissues. Since NO can exert protective or regulatory functions in the cell at a low concentration while toxic effects at higher concentrations, its role may be tightly regulated in the cell. Therefore, this paper was focused on signal transduction pathway of NO synthesis, role of endogenous TGF-$\beta$ in NO production. effect of NO on superoxide formation. Costimulation of murine peritoneal macrophages with interferon-gamma (IFN-γ) and phorbol 12-myristate 13-acetate (PMA) increased both NO secretion and mRNA expression of inducible nitric oxide synthase (iNOS) when PMA abolished costimulation. Pretreatmnet of the cells with PMA abolished costimuation effects due to the depletion of protein kinase C (PKC) activities . The involvement of PKC in NO secretion could be further confirmed by PKC inhibitor, stauroprine, and phorbol ester derivative, phorbol 12,13-didecanoate. Addition of actinomycine D in IFN-γ plus PMA stimulated cells inhibited both NO secretion and mRNA expression of iNOS indication that PMA stabilizes mRNA of iNOS . Exogenous TGF-$\beta$ reduced NO secretion in IFN -γ stimulated murine macrophages. However addition of antisense oligodeoxynucleotide (ODN) to TGF-$\beta$ to this system recovered the ability of NO production and inhibited mRNA expression of TGF-$\beta$. ACAS interactive laser cytometry analysis showed that transportation of FITC -labeled antisense ODN complementary to TGF-$\beta$ mRNA could be observed within 5 min and reached maximal intensity in 30 min in the murine macrophage cells. NO released by activated macrophages inhibits superoxide formation in the same cells . This inhibition nay be related on NO-induced auto -adenosine diphosphate (ADP) -ribosylation . In addition, ADP-ribosylation may be involved in the process of macrophage activation .

• Journal of Microbiology and Biotechnology
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• v.9 no.1
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• pp.113-118
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• 1999

The insulin-like growth factor (IGF) is found in all vertebrates and its type-II molecule is regarded as a fundamental embryonic growth factor during development. We have firstly identified, in this study, a cDNA clone corresponding to IGF-II (flIGF-II) from the adult brain of the teleost, Paralichthys olivaceus. We also examined the tissue expression of flIGF-II in several adult tissues by RT-PCR. The flIGF-II cDNA contained a complete ORF consisting of 215 amino acids and one stop codon. Its molecular characteristics appear to be similar to the previously identified IGF-II molecules, in which a common primary structure exhibiting B, C, A, D, and E domains is evidently observed. This cDNA clone seems to be cleaved at $Ala_{52}$ for the $NH_2$-end signal peptide and appears to produce a 98 amino acid-long E-peptide from the $Arg^{118}$. The functional B-D domain regions, therefore, include 65 amino acids and is able to encode a 7.4-kDa protein. The most prominent structural difference between IGF-I and IGF-II was that the D domain of IGF-II exhibits a two-codon-deleted pattern compared to the 8 amino acid-containing IGF-I. The insulin family signature in the A domain and six cysteins forming three disulfide bridges between the B and A domains were evolutionary-conserved from teleosts to mammalian IGF-II. Interestingly, the E-peptide region appears to provide a distinct hallmark between teleosts in amino acid composition. The flIGF-II shows 85.1% of sequence identity to salmon and trout, 90.6% to tilapia, and 98.4% to perch in amino acid level. In tissue expressions of IGF-II, it is very likely that flIGF-II has a significant expression in the adult brain. However, liver seems to be the main source for IGF-II production, and relatively low signals were observed in the adult muscle and kidney. Taken together, it would be concluded that the functional region for IGF-II mRNA is highly similar in phylogeny and is evolutionary, conserved as a mediator for the growth of vertebrates.

• The Korean Journal of Physiology and Pharmacology
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• v.12 no.4
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• pp.165-170
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• 2008

In the present study, we aimed to identify the synergistic effects of concurrent treatment of low concentrations of cilostazol and probucol to inhibit the oxidative stress with suppression of inflammatory markers in the cultured human coronary artery endothelial cells (HCAECs). Combination of cilostazol (0.3${\sim}3{\mu}$M) with probucol (0.03${\sim}0.3{\mu}$M) significantly suppressed TNF-${\alpha}$-stimulated NAD(P)H-dependent superoxide, lipopolysaccharide (LPS)-induced intracellular reactive oxygen species (ROS) production and TNF-${\alpha}$ release in comparison with probucol or cilostazol alone. The combination of cilostazol (0.3${\sim}3{\mu}$M) with probucol (0.1${\sim}0.3{\mu}$M) inhibited the expression of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) more significantly than did the monotherapy with either probucol or cilostazol. In line with these results, combination therapy significantly suppressed monocyte adhesion to endothelial cells. Taken together, it is suggested that the synergistic effectiveness of the combination therapy with cilostazol and probucol may provide a beneficial therapeutic window in preventing atherosclerosis and protecting from cerebral ischemic injury.

• Fisheries and Aquatic Sciences
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• v.9 no.4
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• pp.140-145
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• 2006

We measured activities of the ubiquitous tripeptide non-protein thiol (L-${\gamma}$-glutamyl-L-cysteinyl-glycine), glutathione (GSH), which is believed to playa fundamental role in detoxifying xenobiotics in biological systems, as a metabolic biomarker for benzo(${\alpha}$)pyrene and Aroclor 1254 exposure in the Pacific oyster Crassostrea gigas. Reproductive oysters were exposed to the pollutants for 50 days by the algal vectoring method in which the oysters were fed with concentrated standard algal foods grown in culture media containing Aroclor 1254 (0, 5, 50, 500 ng/g) or benzo(${\alpha}$)pyrene (0, 10, 100, 1,000 ng/g). Both pollutants induced maternal GSH activities in 10 days in a dosage-dependent manner (p<0.05), although Aroclor 1254 was stronger. The pollutant-driven GSH elevation persisted for 20 to 30 days depending on the pollutants and concentrations. Thereafter, a drastic decline in the GSH activity was observed due to metabolic failure, after which the oyster GSH remained at low levels throughout the remainder of the experiment. The pollutant exposures influenced maternal reproductive output in terms of fertilization, hatching, and morphology. These results imply that changes in activity of the GST-catalyzing molecule can be used as an oyster biomarker for Aroclor 1254 and benzo(${\alpha}$)pyrene exposure.

• Nutrition Research and Practice
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• v.13 no.1
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• pp.3-10
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• 2019

BACKGROUND/OBJECTIVES: The $NAD^+$ precursor nicotinamide riboside (NR) is a type of vitamin $B_3$ found in cow's milk and yeast-containing food products such as beer. Recent studies suggested that NR prevents hearing loss, high-fat diet-induced obesity, Alzheimer's disease, and mitochondrial myopathy. The objective of this study was to investigate the effects of NR on inflammation and mitochondrial biogenesis in AML12 mouse hepatocytes. MATERIALS/METHODS: A subset of hepatocytes was treated with palmitic acid (PA; $250{\mu}M$) for 48 h to induce hepatocyte steatosis. The hepatocytes were treated with NR ($10{\mu}M$ and 10 mM) for 24 h with and without PA. The cell viability and the levels of sirtuins, inflammatory markers, and mitochondrial markers were analyzed. RESULTS: Cytotoxicity of NR was examined by PrestoBlue assay. Exposure to NR had no effect on cell viability or morphology. Gene expression of sirtuin 1 (Sirt1) and Sirt3 was significantly upregulated by NR in PA-treated hepatocytes. However, Sirt1 activities were increased in hepatocytes treated with low-dose NR. Hepatic pro-inflammatory markers including tumor necrosis factor-alpha and interleukin-6 were decreased in NR-treated cells. NR upregulated anti-inflammatory molecule adiponectin, and, tended to down-regulate hepatokine fetuin-A in PA-treated hepatocytes, suggesting its inverse regulation on these cytokines. NR increased levels of mitochondrial markers including peroxisome proliferator-activated receptor ${\gamma}$ coactivator-$1{\alpha}$, carnitine palmitoyltransferase 1, uncoupling protein 2, transcription factor A, mitochondrial and mitochondrial DNA in PA-treated hepatocytes. CONCLUSIONS: These data demonstrated that NR attenuated hepatic inflammation and increased levels of mitochondrial markers in hepatocytes.

• Journal of Life Science
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• v.21 no.1
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• pp.119-126
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• 2011

There is a growing recognition of the significance of $H_2S$ as a biological signaling molecule involved in vascular and nervous system functions. In mammals, two enzymes in the transsulfuration pathway, cystathionine ${\beta}$-synthase (CBS) and cystathionine ${\gamma}$-lyase (CGL), are believed to be chiefly responsible for $H_2S$ biogenesis. Genetic inborn error of CGL leads to human genetic disease, cystathioninuria, by accumulating cystathionine in the body. This disease is secondarily associated with a wide range of diseases including diabetes insipidus and Down's syndrome. Although the human CGL (hCGL) overexpression is essential for the investigation of its function, structure, reaction specificity, substrate specificity, and protein-protein interactions, there is no clear report concerning optimum overexpression conditions. In this study, we report a detailed analysis of the overexpression conditions of the hCGL using a bacterial system. Maximum overexpression was obtained in conditions of low culture temperature after inducer addition, performing low aeration during overexpression, and using a low concentration inducer (0.1 mM, IPTG) for induction. Expressed hCGL was purified by His-tag affinity column chromatography and confirmed by Western blot using hCGL antibody and enzyme activity analysis. We also report that the His tag with TEV site attached protein exhibits 76% activity for ${\alpha}-{\gamma}$ elimination reaction with L-cystathionine and 88% for ${\alpha}-{\beta}$ elimination reaction with L-cysteine compared to those of wild type hCGL, respectively. His tag with TEV site attached protein also exhibits a 420 nm absorption maximum, which is attributed to the binding cofactor, pyridoxal 5'-phosphate (PLP).

• Korean Journal of Microbiology
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• v.47 no.1
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• pp.14-21
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• 2011

Riboflavin synthase catalyzes the formation of one molecule of each riboflavin and 5-amino-6-ribitylamino-2,4-pyrimidinedione by the transfer of a 4-carbon moiety between two molecules of the substrates, 6,7-dimetyl-8-ribityllumazine. The most remarkable feature is the sequence similarity between the N-terminal half (1-97) and the C-terminal half domain (99-213). To investigate the structure and fluorescent characteristics of the N-terminal half of riboflavin synthase (N-RS) in Escherichia coli, more than 10 mutant genes coding for the mutated N-terminal domain of riboflavin synthase were generated by polymerase chain reaction. The genes coding for the proteins were inserted into pQE vector designed for easy purification of protein by 6X-His tagging system, expressed, and the proteins were purified. Almost all mutated N-terminal domain of riboflavin synthases bind to 6,7-dimethyl-8-ribityllumazine and riboflavin as fluorescent ligands. However, N-RS C47D and N-RS ET66,67DQ mutant proteins show colorless, indicating that fluorescent ligands were dissociated during purification. In addition, most mutated proteins show low fluorescent intensity comparing to N-RS wild type, whereas N-RS C48S posses stronger fluorescent intensity than that of wild type protein. Based on this result, N-RS C48S can be used as the tool for high throughput screening system for searching for the compound with inhibitory effect for the riboflavin synthase.